The goal of the proposed research is to isolate and characterize a stable, highly folded form of rat liver mitochondrial DNA. The features of this new physical form are consistent with current concepts of the compact in vivo state of the mitochondria genome. That is, the isolated structure contains a densely folded, covalently closed circular DNA which is packaged into a discrete entity by non-DNA constituents. Most notable among these tightly bound components is an array of proteins some of which may be derived from the inner mitochondrial membrane. It is the specific intent of this proposed study to establish the total biochemical constituency and the molecular architecture of the packaged mtDNA. The role of the bound constitutents in the stabilization and function of the folded genome will be examined in depth. The folded DNA will be released from isolated mitochondria by gentle lysis and fractionated on sucrose density gradients and metrizamide density gradients. The structural integrity of the particle will be monitored by electron microscopy. The variety of bound constituents will be established by chemical and biochemical means and quantified relative to the content of DNA. The proteins from various fractions will be analyzed on the basis of size and ionic properties in SDS-and isoelectric focusing polyacrylamide gels and their spacial arrangement, relative to each other and to the DNA, will be examined by specific crosslinking experiments. The amino acid composition of those polypeptides which play a major role in the maintenance of the folded structure will be determined. Localization of the sub-cellular site of biosynthesis of the individual bound proteins will be carried out in radio-labeling experiments in vivo in the presence and absence of specific translation inhibitors. The size classes of nascent DNA strands contained in the various supramolecular forms of the folded DNA will be examined by electrophoresis of the dissociated fragments in agarose-polyacylamide composite gels. Finally, the specific sequence of mitochondrial DNA involved in protein recognition and binding will be probed with the aid of restriction endonucleases.